plasmid extraction - middle tennessee state university：质粒提取-田纳西中部州立大学

《plasmid extraction - middle tennessee state university：质粒提取-田纳西中部州立大学》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、Genetics3250SequenceAnalysisCahoon–Genetics3250LabMiddleTennesseeStateUniversityChloroplastGenomeProject–Week1Inplantcells,DNAcanbefoundinthreeorganelles–nuclei,chloroplasts,andmitochondria.nucleuschloroplastmitochondrionmitochondrionchloroplastchloroplastThechloroplastisthesite

2、ofphotosynthesisinplantcells.It’sgenomeanditsprocessofgeneexpressionisverysimilartothosefoundinbacteria.Maieretal.1995,J.Mol.Biol251:614-628ThechloroplastgenomecanbeimaginedasacircularpieceofDNAabout120,000–140,000basesinsizewithapproximately100genes.Thisdiagramrepresentsthecorn

3、chloroplastgenomewhichwascompletedin1995.4Genetics3250SequenceAnalysisCahoon–Genetics3250LabMiddleTennesseeStateUniversityYouandyourgeneticsclasscolleaguesareattemptingtosequencethechloroplastgenomeoftallfescue(Festucaarundinacea).Intactgenomesaregenerallytoolargetosequence.Toge

4、taroundthisproblemagenomemustbecutintolotsofsmallmanageablepieces.Thissemester,twoindependentstudystudentshaveisolatedthechloroplastgenome,mechanicallyshearedtheDNAintosmallpiecesandthenplacedthemintoplasmidcloningvectorsforsequencing.genomePiecesputintoplasmidvectorsShearingfor

5、ceGenomecutintothousandsoftinyrandompiecesTheseplasmidsarethengrowninsidebacteriathatwillproducethemillionsofcopiesthatarerequiredformanipulationandsequencing.Todayyouwillextractaplasmidfromabacterialculture.Nextweekwewillusethisplasmidtosetupasequencingreaction.PlasmidDNAExtrac

6、tionPlasmidExtractionGoal–extracttheplasmidDNAfromaliquidbacterialculture.Materials·ResuspensionBuffer(P1)·LysisBuffer(P2)·NeutralizationBuffer(N3)·WashBuffer(PE)·SpinColumn·ElutionBuffer(EB)·Collectiontubes4Genetics3250SequenceAnalysisCahoon–Genetics3250LabMiddleTennesseeStateU

7、niversityIntroductionTodayyouwilluseacommercialkit(Qiagen’sMiniprep)toextractandpurifyplasmidDNAfromyourbacterialculture.PlasmidsaresmallcircularpiecesofDNAthataremaintainedandreplicatedseparatelyfromthebacterialcell’sgenome.Procedure1.Pipetteorpour2.0mlofcultureintoacoloredmicr

8、ofugetube.2.Pelletcellsfor1minuteatfullspeed.3.